Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad, Scaphiopus couchii

Both pyruvate kinase (PK) and phosphofructokinase (PFK) occur in two differ ent forms, separable by isoelectric focusing (IEF), in skeletal muscle of t he spadefoot toad Scaphiopus couchii. During estivation (aerobic dormancy) the proportions of the two forms changed compared with controls; in both ca ses the amount of enzyme in Peak I (pI = 5.3-5.4) decreased whereas activit y in Peak II (isoelectric point = 6.2-6.4) increased. In vitro incubation o f crude muscle extracts with P-32-ATP under conditions that promoted the ac tivity of cAMP-dependent protein kinase led to strong radiolabeling associa ted with Peak I, but not Peak II, and reverse phase HPLC confirmed that P-3 2 was associated with the subunits of both PK and PFK found in Peak I. Spec ific radiolabeling of Peak I PK and PFK by protein kinase A was further con firmed using immunoprecipitation. In total, this information allowed identi fication of the Peaks I and II enzymes as the phosphorylated and dephosphor ylated forms, respectively, and the effect of estivation was to increase th e proportion of dephosphorylated PK and PFK in muscle. Analysis of the kine tic properties of partially purified PK and PFK revealed significant kineti c differences between the two forms of each enzyme. For PK, the Peak II (lo w phosphate) enzyme showed a 1.6-fold higher Km for phosphoenolpyruvate and a 2.4-fold higher Ka for fructose-1,6-bisphosphate than did the Peak I (hi gh phosphate) form. These kinetic properties suggest that Peak II PK is the less active form, and coupled with the shift to predominantly the Peak II form during estivation (87% Peak II vs. 13% Peak I), are consistent with a suppression of PK activity in estivating muscle, as part of the overall met abolic rate depression of the estivating state. A similar shift to predomin antly the Peak II, low phosphate, form of PFK (75% Peak II, 25% Peak I) in muscle of estivating animals is also consistent with metabolic suppression since phosphorylation of vertebrate skeletal muscle PFK is typically stimul ated during exercise to enhance enzyme binding to myofibrils in active musc le. Peak II PFK also showed reduced sensitivity to inhibition by Mg:ATP (I5 0 50% higher) compared with the Peak I form suggesting that the enzyme in e stivating muscle is less tightly regulated by cellular adenylate status tha n in awake toads. The data indicate that reversible phosphorylation control over the activity states of enzymes of intermediary metabolism is an impor tant mechanism for regulating transitions between dormant and active states in estivating species.